Q Beta (Q.beta.) replicase is a template specific RNA directed RNA polymerase derived from the bacteriophage Q.beta.. In vivo, the normal function of Q.beta. replicase is to replicate the RNA genome of the Q.beta. bacteriophage to produce progeny phage genomes. The Q.beta. virion contains one molecule of single stranded RNA which is termed the viral plus strand. This is a strand utilized as mRNA to direct viral protein synthesis. The Q.beta. replicase enzyme uses this plus strand as the template to produce RNA copies which are complementary to the original template. These RNA molecules are termed minus strands.
Importantly, both the plus and minus strands are templates for the enzyme. Therefore, the replication of the RNA template proceeds in an exponential fashion. Thus, a few molecules of the replicatable RNA template are replicated in an exponential fashion so long as the enzyme is present in molar excess.
The enzyme also has the capacity to replicate certain RNA and DNA templates in vitro. One well studied example of such an RNA template is the MDV-1 RNA (Mills et al., Science 180:916-927 (1973)). The amplification of MDV-1 DNA by the enzyme Q.beta. replicase has been disclosed, for example in published European Patent Application 91309430.6 (publication number 0 481 704 A1). Under conditions appropriate for replication, each round of replication takes approximately 15 seconds. Under these conditions, a single MDV-1 molecule will yield 10.sup.12 progeny strands in only thirteen minutes (Pritchard and Stefano, Ann. Biol. Clin. 48:492-497 (1990)). This is an amount of RNA which is easily detectable by conventional methods such as fluorescence.
Additional nucleic acid sequences may be joined to the Q.beta. replicase template sequences to generate a recombinant template which is useful as a hybridization probe for the detection of a target nucleic acid sequence in a sample. In general, the additional nucleic acid sequence can be embedded at tolerant positions within the replicatable RNA or DNA sequence (see e.g., Miele et al., J. Mol. Biol. 171: 281-295 (1983)), or it can be added to either the 5' or 3' end of the replicatable template molecule (see e.g., U.S. Pat. No. 5,112,734). Either of these embodiments are useful in conventional hybridization methods.
Typically, such hybridization methods require that nucleic acids from an experimental sample be fixed to a solid support (e.g., nitrocellulose or nylon membranes). The replicatable nucleic acid sequence, having the additional probe sequence attached, is contacted with the support bound nucleic acids under conditions appropriate for hybridization. After removing non-specifically bound nucleic acid by conventional methods, the Q.beta. replicase system is used to amplify specifically bound replicatable template sequences. The replication of the template molecule in the sample is an indication of the presence of the target nucleic acid in the sample.
The sensitivity of this system, however, is limited due to the generation of "background signal" or "false positives". Typically, preparations of Q.beta. replicase contain endogenous replicatable RNA species. When such a preparation of Q.beta. replicase is incubated near neutral pH in the presence of nucleoside triphosphates and magnesium ions, but without exogenously added template RNA, the endogenous replicatable RNA species is amplified exponentially. Using commonly employed detection methods (e.g. fluorescence), this amplified endogenous species would be indistinguishable from the amplified product of a recombinant template of the type described above. The detection of this endogenous species in a hybridization assay in which an exogenous RNA template containing a probe sequence has been added to detect the presence of a target nucleic acid sequence results in the identification of false positives.
The endogenous replicatable RNA species which gives rise to the false positives is extremely difficult to completely remove in the process of preparing a Q.beta. replicase enzyme batch. Although methods have been reported which can be used to prepare an enzyme batch which does not contain an endogenous species (see e.g., U.S. Ser. No. 07/364,306, filed on Jun. 9, 1989, even such methods are known to exhibit batch to batch variability with respect to the endogenous species.
In addition to Q.beta. replicase, other enzymes are known to have an RNA dependent RNA polymerase activity. For example, the DNA dependent RNA polymerase from coliphage T7 is known to replicate certain small RNA molecules in a manner analogous to Q.beta. replicase (see e.g., Konarska and Sharp, Cell 63:609-618 (1990)). Application of T7 RNA polymerase to assays similar to those described above is very likely to involve analogous false positive problems. Indeed it is possible that the known RNA substrates for T7 RNA polymerase arose originally in cells which were a source of the enzyme and that these or other such substrates could give rise to false positive results in assays based on RNA amplification by T7 RNA polymerase (or any other enzyme having an RNA dependent RNA polymerase activity). A method for addressing this problem of false positives in an amplification assay would represent a clear advance in the art.